Noi riconosciamo le donazioni tipo di ceppi batterici dai gruppi del Prof. Judith Armitage (Università di Oxford, UK) e il Prof. Corrado Mullineaux (Queen Mary University of London, UK). IMD è finanziato congiuntamente dal Dipartimento di Biochimica (Oxford University) e OCISB; AR è finanziato da un Engineering and Physical Sciences Research Council (EPSRC) borsa di studio DTC; ND è finanziata dal Biotechnology and Biological Sciences Research Council (BBSRC); MCL è finanziato da una borsa di studio della Royal Society University Research.

<ol><li> Per iniziare questa procedura, 50 ml di scorte congelate della proteina fluorescente che esprimono Escherichia coli cellule batteriche vengono prima scongelate e coltivate aerobicamente con agitazione in 5 ml di LB mezzi di crescita durante la notte a 37 ° C. Al mattino, 50 ml di questa cultura satura viene estratto e sub-coltura in mezzi di glucosio minima M63 cultura, incubando a 30 gradi C per 4 a 6 ore. Qui mostriamo che utilizza due differenti ceppi cellulari, uno dei quali esprime un elettrone-trasporto citocromo fuso con GFP, l&#39;altra che esprime una proteina coinvolta nel motore flagellare batterico fuso a GFP. </li><li> Le cellule possono sia raccolto direttamente dal crescente sub-cultura, se loro visione come campioni immobilizzati, o possono essere tosato per troncare flagelli batterici se la visualizzazione in &quot;legato&quot; le condizioni. </li><li> Tosatura prevede il posizionamento di solito 1-5ml di sub-cultura in un dispositivo costituito da due siringhe sterili uniti da tubi sterili. La tosatura viene effettuata alternando spingendo in ogni pompa a siringa per spingere la cultura attraverso il tubo stretto. Questo viene fatto 50-100 volte, a seconda della entità del taglio richiesto. La cultura viene poi centrifugato per agglomerare le cellule, che è risospeso in terreno minimo da rimuovere frammenti flagelli. </li><li> Abbiamo poi preparare pulito BK7 coprioggetto di vetro immergendo in una soluzione satura di KOH in etanolo per 20 minuti, poi sciacquare accuratamente in acqua deionizzata e etanolo e lasciare asciugare all&#39;aria per almeno 1 h. </li><li> Costruiamo un semplice flusso di celle per ospitare le cellule al microscopio. Questa consiste nel definire le linee di grasso paraffina su un vetrino da microscopio BK7 vetro e quindi la creazione di un tunnel-panino mettendo uno dei coverlips pulita in cima, e premendo delicatamente con un paio di pinze, dando un volume di cella di flusso di 5-10 microlitri . </li><li> Per osservare le cellule immobilizzate riempiamo il flusso delle cellule da iniezione con una w / v soluzione 0,1% di poli-L-lisina, e permettono di incubare a temperatura ambiente per almeno 1 minuto. Abbiamo poi scovare con 100 microlitri mezzi minimi, iniettando i media da un capo all&#39;altro del flusso delle cellule e che la migrazione con carta velina dall&#39;altro. </li><li> Abbiamo poi lo stoppino attraverso 20 ml di una diluizione 1:500 di 200 nm di diametro in lattice microsfere (Polysciences) in terreno minimo per contrassegnare la superficie coprioggetto. Il flusso delle cellule è invertito in modo che il coprioggetto sia rivolto verso il basso, posto su una piattaforma chiara della superficie in una camera umida semplice, e incubate a temperatura ambiente per 5 min. Perline legato sono poi lavate via dalle traspirazione a 100 microlitri mezzi minimi. </li><li> Se vogliamo osservare le cellule legato omettiamo la poli-L-lisina passo e invece riempire la cella a flusso con 5 mg / ml di anticorpi anti-flagellina. Il flusso delle cellule è posto nella camera umida per 10 minuti e viene poi eliminata attraverso di traspirazione. </li><li> 20 l di la coltura cellulare viene poi malvagio attraverso il flusso delle cellule, sia utilizzando il campione tranciati se osservando le cellule legato, incatenato o il campione unsheared nella visione cellule immobilizzate. </li><li> Il flusso delle cellule è invertito e collocato nella camera umida per 20 minuti. Le cellule non legati vengono lavati da traspirazione a 100 microlitri mezzi minimi. </li><li> Una goccia di olio di immersione si trova nel centro della superficie superiore del vetrino e il flusso delle cellule viene poi messo gentilmente a supporto del campione del costume microscopio a fluorescenza costruito, il contatto ottico con le lenti ad alta apertura numerica dell&#39;obiettivo. </li><li> L&#39;elettrone-moltiplicando microscopio fotocamera è accesa e la fotocamera impostata per essere raffreddato a -70 ° C, il software è impostato per acquisire immagini ad un frame rate tipico di 25 Hz in frame-modalità di trasferimento, inizialmente disenabling il controllo di guadagno sul fotocamera. </li><li> L&#39;illuminazione campo chiaro è acceso e l&#39;immagine viene messa a fuoco, la selezione di un cellulare adeguata o di un gruppo di cellule da acquisire sulla base del loro essere bloccato con le loro asse parallelo alla superficie lungo coprioggetto. L&#39;attenzione è finemente regolata per garantire che i 200 sfere di lattice nm attaccato alla superficie coprioggetto sono solo a fuoco. </li><li> Una sequenza di immagini viene acquisita in campo chiaro per registrare il contorno del corpo cellulare. L&#39;illuminazione campo chiaro è spento e il guadagno della telecamera è abilitato al massimo. </li><li> Per un totale di acquisizione standard utilizzando la fluorescenza di riflessione interna (o TIRF) un fascio laser sulla lunghezza d&#39;onda appropriata (in questo caso l&#39;eccitazione proteina fluorescente verde, si usa un laser a 473 nm) è pre-impostato in modo da essere concentrati nel piano focale posteriore della la lente dell&#39;obiettivo, ma spostato lateralmente rispetto all&#39;asse ottico per generare un campo evanescente per l&#39;eccitazione di fluorescenza nel campione di cellule. </li><li> L&#39;acquisizione della fotocamera viene avviato e l&#39;otturatore aperto laser per eccitare le proteine ​​fluorescenti all&#39;interno del batterio. I parametri per l&#39;intensità del laser e la velocità di acquisizione devono essere ottimizzati per il particolare sistema biologico sotto inchiesta da sperimentare con valori diversi, ma una gamma tipica rignificative a studiare complessi proteici mobile nella membrana cellulare sono 10-10 mW di potenza del laser su una superficie di eccitazione circolare del diametro di circa 30 micron, con un tempo di esposizione per ogni fotogramma di 50-40 ms. I campioni sono illuminate fino fotodecolorate, di solito per circa 10 s. </li><li> Questo protocollo può essere utilizzato sia per le cellule legato in cui il corpo della cellula ruota su un punto di attacco tra il coprioggetti e un mozzicone di flagelli, e per le cellule immobilizzate che sono rigidamente fissate alla superficie coprioggetto. </li><li> Alcuni ceppi di cellule che hanno un elevato numero di complessi copia beneficiare di una prima diffrazione limitata candeggina laser focalizzato su un polo della cellula prima TIRF imaging. Questa candeggina è equivalente a quella utilizzata per il recupero di fluorescenza dopo photobleaching (o FRAP). Utilizzando il nostro microscopio personalizzati parte della luce laser di eccitazione può essere alimentata via in un secondo percorso indipendente utilizzato per FRAP-tipo sbiancamento. Tipicamente 1-10mW potenza del laser è utilizzato, con un tempo di candeggina tipico nella gamma 10-300 ms. Il risultato è in contrasto con immagini molto più alta nella zona sbiancata della cellula permettendo complessi individuali che successivamente diffusa in quella zona per essere più facili da visualizzare. </li><li> Per visualizzare i complessi nel citoplasma cellulare che diffondono molto più velocemente rispetto a quelli della membrana cellulare è impiegato un modo diverso di illuminazione chiamato &quot;slimfield&quot;. Ecco un laser focalizzato si espande lateralmente fino a comprendere solo una singola cella. Questo produce un campo molto intenso che permette esposizioni molto più veloce di solito un millisecondo da adottare. </li><li> A seguito di acquisizione dati, le immagini sono inseriti in software personalizzato scritto (codificato in LabVIEW 8.5). Questo rileva automaticamente la posizione delle macchie fluorescenti nelle cellule con una precisione di pochi nanometri tipicamente ed estratti delle loro dimensioni e luminosità. Il brightnessof la traccia photobleaching rispetto al tempo di un complesso molecolare tracciato viene quindi utilizzato per stimare la stechiometria, cioè quante singole proteine ​​fluorescenti formano un unico complesso molecolare. </li></ol> Rappresentante dei risultati: Quando il protocollo è fatto correttamente le immagini delle cellule visto in campo chiaro è molto distinto, con i perimetri dei corpi cella buia contro un corpo cellulare bianco / grigio (Figura 1a). In fluorescenza utilizzando cellule immobilizzate, possiamo vedere l&#39;intensità distinte macchie, di solito 250-300 nm di larghezza (figura 1b). Sane, le cellule si vedrà legato a ruotare attorno al punto di attacco legare in campo chiaro le immagini. Sotto eccitazione di fluorescenza alcuni complessi molecolari nel nostro caso potrebbe anche essere visto al punto di attacco, che indica una localizzazione della proteina taggati con il motore flagellare. Queste macchie sono individuali complessi molecolari e il numero di viste dipende dalla modalità di illuminazione utilizzati e quanti dei complessi sono in realtà presenti nella cellula in un dato momento. La mobilità dei punti dipende dal sistema biologico specifico oggetto di studio. Se la densità dei punti è inizialmente molto alta, come è il caso con i citocromi marcato usato qui, poi eseguire una candeggina FRAP iniziale può migliorare il contrasto delle immagini. <img src="/files/ftp_upload/1508/1508Figure1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. (A) Brightfield e (B) immagine TIRF (falsi colori) per un immobilizzato cellule di Escherichia coli che esprimono una proteina fusa alla proteina fluorescente verde (GFP) che è noto per essere coinvolto nei motori flagellare di batteri. Si prega di <a href="https://www.jove.com/files/ftp_upload/1508/1508Figure1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1.

<ol>
	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

The authors have nothing to disclose.

Bisogna fare attenzione a non &quot;oltre al taglio&quot; delle cellule per guardare i batteri legati, poiché questo può compromettere la funzionalità del motore flagellare. E &#39;importante utilizzare le cellule per molto più tempo di un&#39;ora una volta sul vetrino da microscopio in quanto possono diventare ossigeno scarica. Notevole ottimizzazione può essere richiesto di trovare migliori condizioni di imaging microscopio soddisfatti al vostro specifico sistema biologico sotto inchiesta. Potrebbe essere saggio ...

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

Visione completa dei meccanismi delle cellule viventi può essere raggiunto solo attraverso lo studio dei processi chiave che suscitare e dirigere gli eventi a livello cellulare. Fino ad oggi la complessità dei sistemi biologici taglio ha causato preciso singola molecola sperimentazione di essere troppo esigente, concentrandosi invece su studi di singoli sistemi con massa relativamente greggio insieme alla media delle misurazioni. Tuttavia, molti processi importanti si verificano nella cellula vivente a livello di solo uno o poche molecole; misure ensemble generalmente mascherare la natura stocastica ed eterogenea di questi eventi. Qui, utilizzando avanzate di microscopia ottica e strumenti di analisi di analisi delle immagini ci mostrano come monitorare le proteine ​​all&#39;interno della cellula batterica vivente solo con una precisione di singole molecole e come possiamo osservare la dinamica all&#39;interno di complessi molecolari del funzionamento macchine biologiche. Le tecniche sono direttamente rilevanti fisiologicamente. Si tratta di mini-perturbativa e non-invasivo per il campione biologico oggetto di studio e sono completamente in sintonia per le indagini in materia viva, caratteristiche non facilmente disponibili ad altri approcci singola molecola di biofisica. Inoltre, i campioni biologici studiati tutti i prodotti proteina fluorescente-tag a livelli che sono quasi identici ai ceppi cellulari non modificato (&quot;codifica genomica&quot;), in contrasto con l&#39;approccio più comune ma meno ideale per la produzione di proteine ​​molto più di quanto avverrebbe naturalmente (&#39;plasmide di espressione&#39;). Così, i campioni biologici reali, che saranno oggetto di indagine sono molto più vicini al organismi naturali, e quindi le osservazioni più rilevanti per le reali processi fisiologici.

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Qui mostriamo i protocolli per l&#39;esecuzione di singola molecola microscopia a fluorescenza a vivere cellule batteriche per permettere funzionali complessi molecolari per essere rilevati, monitorati e quantificati.

Visualizzazione singola complessi molecolari In Vivo Utilizzo avanzato microscopia a fluorescenza

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

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Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

9.2K Views.  University of Oxford. Qui mostriamo i protocolli per l'esecuzione di singola molecola microscopia a fluorescenza a vivere cellule batteriche per permettere funzionali complessi molecolari per essere rilevati, monitorati e quantificati.

Video: Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Ricerca

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualizzare batteri nei nematodi usando la microscopia a fluorescenza

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Analisi quantitativa di autofagia usando 3D avanzato microscopia a fluorescenza

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

Imaging funzionale delle fabbriche di trascrizione virale utilizzando la microscopia a fluorescenza 3D

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualizzazione della struttura dei linfonodi e della localizzazione cellulare mediante la microscopia confocale Ex-Vivo

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...